1. Field of Invention
The present invention relates to radioimmunoassay and, more particularly, to use of a 96-welled micro-plate in radioimmunoassay.
2. Related Prior Art
Radioimmunoassay (“RIA”) was developed by Yalow and Berson in 1959 for measuring the concentration of the insulin in the blood of a diabetic patient. In typical radioimmunoassay, a target material to be measured is used as an antigen for producing an antibody. The antibody is attached to a test tube. A purified standard of the target material is labeled with iodine-125 as a tracer. A sample of the target material and the labeled standard of the target material are filled in the test tube for incubation. The target material in the sample and the standard of the target material labeled with the iodine-125 compete against each other to bind to the antibody attached to the test tube. If the concentration of the target material in sample is high, the binding amount of the standard labeled with the iodine-125 is low. Therefore, in radioactive counting, the value is low if the concentration of the target material in the sample is high, and the value is high if the concentration of the target material in the sample is low.
The typical radioimmunoassay is often conducted in a single test tube as discussed above. A layer of an antibody is coated on an internal side of the test tube. A sample of a target material is filled in the test tube for incubation so that the antibody binds the target material in the sample. Other materials are washed away after the target material is binding to the antibody. Then, the standard of the target material is labeled with a radionuclide and filled in the test tube for competition with the target material. Later, an excessive portion of the sample of the target material labeled with the radionuclide is washed away. The concentration of the target material in the sample is learned from the radioactive reading in radioactive counting. The radionuclide is generally iodine-125 and the radioactive counting is therefore based on gamma ray. However, gamma ray will penetrate, and therefore a reader can only be used to read only one test tube. To use a single test tube in such an experiment is not without any problem. As radioimmunoassay was developed long time ago, and only a few automatic or semiautomatic instruments have been devised since. The price of such an automatic or semiautomatic instrument is expensive and imposes a heavy financial burden on a user. To operate the tube manually without using any automatic or semiautomatic instrument is however limiting regarding the rate of the handling of samples. On the other hand, should a 96-welled micro-plate be used in an experiment, the radioactive reading of the gamma ray in a well would be interfered with by the gamma ray in another well.
There has been an attempt to use a multi-welled micro-plate in typical radioimmunoassay. Small plastic balls are coated with an antibody before they are located in the multi-welled micro-plate for incubation. After the incubation, the small plastic balls are transferring to test tubes for radioactive counting. This method has rarely been used, and related instruments are also rare and expensive.
If the nuclide that radiates gamma ray is replaced with a nuclide that radiates beta ray such as 3H for radioimmunoassay, a 96-welled micro-plate can be used. Accordingly, liquid scintillation analysis is used. Beta ray is not strong and does not penetrate the wells of the 96-welled micro-plate which is generally made of a plastic material. Hence, the radioactive reading of the beta ray in a well is not interfered with by the beta ray in another well. In the radioactive counting, scintillation liquid is filled in the wells and reacted with the beta ray so that photons are emitted. A liquid scintillation analyzer related to the 96-welled micro-plate is used to measure the readings of the beta ray in the wells. The use of a beta ray-radiating nuclide in radioimmunoassay is however not popular in comparison with iodine-125.
There has been developed another method that can be used to measure the concentration of the insulin in the blood of a diabetic patient, i.e., immunoradiometric assay (“IRMA”). In typical immunoradiometric assay, a target material to be measured is used as an antigen for producing capture antibody and detection antibody. The two antibodies recognize different epitopes of the antigen. At first, the capture antibody is attached to a test tube while the detection antibody is labeled with iodine-125. Later, a sample of the target material (antigen) is filled in the test tube for incubation. Now, the antigen in the sample is binding to the capture antibody in the test tube. Then, the detection antibody is filled in the test tube. The detection antibody recognizes the antigen binding to capture antibody. In radioactive counting, the concentration of the target material in the sample is high as the amount of the detection antibody binding in the test tube is large. Therefore, the concentration of the target material in the sample is in positive proportion with the radioactive reading. This method is sometimes called the “sandwich method.”
The present invention is therefore intended to obviate or at least alleviate the problems encountered in prior art.